MALDI Imaging Mass Spectrometry Nan Kleinholz Mass Spectrometry and Proteomics Facility The Ohio State University Mass Spectrometry and Proteomics Workshop
What is MALDI Imaging? MALDI: Matrix Assisted Laser Desorption Ionization Imaging: Imaging is a technique in which a sample, often a thin tissue section, is moved in two dimensions while the mass spectrum is recorded.
Things to think about Goals Small molecule/lipids Peptide/protein Others Sample type Heart, lung, liver, etc. Biofilm Others Method optimization! Appropriate matrix Standards
IMS Workflow Sample Preparation Matrix Application Analysis Processing
Sample Preparation Fresh Frozen (FF) is best for all IMS experiments. BUT: Hundreds and thousands of tissue samples have been banked over the years as Formalin Fixed Paraffin Embedded (FFPE). Lipids and small molecules: FF only. Intact proteins: FF or FFPE
FFPE samples: Tissue sectioning Deparaffinization and Rehydration Heat induced antigen retrieval Histology staining on serial section (i.e. Hematoxylin and eosin) Matrix application FF samples: Tissue sectioning Washing Histology staining on serial section Matrix application
Microtome-FFPE Cryostat-FF
Matrix Application Wet, but not too wet. Extraction Delocalization Sufficient matrix application. Some But not too much Matrix choice Works well Doesn t work And the list goes on
Automated Matrix Application Methods Spotted Arrays Labcyte Portrait uses an acoustic pico drop method
Bruker ImagePrep HTX Technologies Sprayer TM-Sprayer
Manual Matrix Application Methods TLC sprayer Sublimation
Typical Workflow for peptide and protein imaging Tissue slice on slide Wash tissue by pipette or bath Common solvents: Ethanol Acetonitrile Apply trypsin for digestion Incubate at 37 C for 2-18 hours Allow tissue to dry Apply matrix SA, DHA: proteins HCCA, DHB: peptides MALDI IMS
Top-Down or Bottom-Up Analysis? Top-Down: extract and ID intact proteins in images Faster sample prep for imaging Fewer steps means less chance for delocalization or other error Increase confidence in ID Bottom-Up: sequence tryptic fragments of larger proteins Easier to analyze larger or hydrophobic proteins Potential for MALDI MS/MS
Top Down Analysis for intact proteins Tools for Protein ID post IMS Bottom-Up Analysis for tryptic peptides Matrix removal Matrix removal Tissue microextraction Tissue microextraction LC-MS of proteins LC-MS of peptides ETD MS/MS fragmentation CID MS/MS fragmentation De-novo sequencing Match masses of protein ID s to IMS data Database searching
Typical workflow for lipids and small molecule imaging Fresh Frozen tissue samples Wash with 50mM Ammonium formate to reduce salts Lipids: DHB, DHA, DAN, 9-AA Apply appropriate matrix Small molecules: DHB, SA, HCCA, THAP MALDI IMS
Analysis Instrumentation in our facility with imaging capability Bruker ultraflextreme MALDI-TOF-TOF Bruker solarix FTMS MALDI and ESI
Nature Reviews Cancer 10, 639-646 (September 2010) doi:10.1038/nrc2917
Processing A challenge in imaging experiments is the huge amount of generated data. A typical MALDI imaging dataset contains thousands of spectra. Software programs allow statistical analysis and overlaying of the histology staining. (SCiLS Bruker supported.)
H and E staining with tumor indicated Dataset provided by SCiLS
Total mass spectrum for entire imaging run
Three masses selected as having statistical differences
Wrapping it all up MALDI IMS is a rapidly growing field within mass spectrometry. MALDI IMS is providing major contributions to the understanding of diseases, improving diagnostics, and drug delivery. MALDI IMS is time consuming and involves lots of sample manipulation and data interpretation. (It is not one size fits all.)
For further information, please come see me in BRT 250. Not actually me